Summary G protein-coupled receptors (GOCRs) regulate a variety of cell functions and are important targets of therapeutics. A fundamental but poorly understood question in studying GPCRs is how targeted GPCR trafficking is achieved. The overall goal of our research program is to elucidate the molecular mechanisms of nascent GPCR targeting to their functional destinations and to understand the role of targeting in modulating cellular responses to hormones and drugs. Under this broad objective, this proposal will use ?2- adrenergic receptors (?2-ARs) as models to address the following two questions which are central to understanding GPCR anterograde trafficking: 1) how newly synthesized GPCRs export from the endoplasmic reticulum (ER) and then transport en route from the ER through the Golgi to the cell surface; and 2) how GPCRs are sorted from one another and from non-GPCR plasma membrane proteins during their traffic along the biosynthetic pathway. The premise of this project is our recent findings that the ER- Golgi-cell surface transport and sorting of ?2A-AR and ?2B-AR, two closely related, prototypic GPCRs, are coordinated by ufmylation, an ubiquitination-like post-translational modification, and three interacting proteins, C1orf27 (an ER membrane protein), coiled-coil ?-helical rod protein 1 (HCR1) and Golgi-localized ? ear-containing ARF-binding protein 3 (GGA3). We will define the novel functions of protein ufmylation and C1orf27 in the ER-Golgi transport and sorting, as well as HCR1 and GGA3 in the post-Golgi traffic and sorting of ?2A-AR and ?2B-AR, and elucidate the underlying mechanisms. The proposed research is a continuation of our long-standing efforts to study the anterograde trafficking of GPCRs, which have led to the discovery of a number of structural determinants and regulatory proteins essential for GPCR export and sorting. As in the past, we will employ state-of-the-art biochemical, immunochemical and live cell imaging techniques to dissect the mechanistic aspects of GPCR trafficking along the biosynthetic pathway, including maturation, sorting and targeting. These studies will provide important insights into regulation of GPCR export trafficking which is a poorly explored area in the study of the GPCR superfamily.